Method for culturing skeletal muscle for tissue engineering

ABSTRACT

The invention provides a nutrient medium composition and associated methods for lengthening the useful life of a culture of muscle cells. Disclosed is a method of culturing mammalian muscle cells, including preparing one or more carriers coated with a covalently bonded monolayer of trimethoxy-silylpropyl-diethylenetriamine (DETA); verifying DETA monolayer formation by one or more associated optical parameters; suspending isolated fetal rat skeletal muscle cells in serum-free medium according to medium composition 1; plating the suspended cells onto the prepared carriers at a predetermined density; leaving the carriers undisturbed for cells to adhere to the DETA monolayer; covering the carriers with a mixture of medium 1 and medium 2; and incubating. A cell nutrient medium composition includes Neurobasal, an antibiotic-antimycotic composition, cholesterol, human TNF-alpha, PDGF BB, vasoactive intestinal peptides, insulin-like growth factor 1, NAP, r-Apolipoprotein E2, purified mouse Laminin, beta amyloid, human tenascin-C protein, rr-Sonic hedgehog Shh N-terminal, and rr-Agrin C terminal.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.12/765,399, filed Apr. 22, 2010, which claims priority to U.S.Provisional Application No. 61/171,968, filed Apr 23, 2009, which arehereby incorporated by reference in their entirety.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with Government support, under contract/grantnumber R01 NS050452 awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to the field of tissue engineering and,more particularly, to a system and method of extending the in vitrouseful life of a culture of muscle cells.

BACKGROUND OF THE INVENTION

Skeletal muscle differentiation and maturation is a complex processinvolving the synergy of different growth factors and hormonesinteracting over a broad time period [1-11]. The differentiation processis further complicated by neuronal innervation, where neuron to musclecell signaling can regulate myosin heavy chain (MHC) gene expression andacetylcholine receptor clustering [12-18]. Consequently, understandingof the role of the growth factors, hormones and cellular interactions inskeletal muscle differentiation would be a key step in generatingphysiologically relevant tissue engineering constructs, developingadvanced strategies for regenerative medicine and integrating functionalskeletal muscle with bio-hybrid MEMS devices for non-invasiveinterrogation in high-throughput screening technologies.

In order for skeletal muscle myotubes developed in vitro to be useful intissue engineering applications, they must exhibit as many of thefunctional characteristics of in vivo skeletal muscle fibers aspossible. During muscle fiber development in vivo, several criticalstructural changes occur that indicate functional maturation of theextrafusal myotubes. These changes include sarcomere organization,clustering and colocalization of ryanodine (RyR) and dihydropyridine(DHPR) receptors and MHC class switching [19-23]. Each of thesestructural changes reflects the physiological maturation of the skeletalmuscle and is critical for consistent muscular contraction. For example,organization of the contractile proteins myosin and actin intosarcomeric units gives skeletal muscle myotubes organized and structuredcontraction, a property lacking in smooth muscle. The organization ofsarcomeres in skeletal muscle gives rise to anisotropic and isotropicbands of proteins (A and I bands) and gives skeletal muscle a striatedappearance. The clustering and colocalization of RyR and DHPR isindicative of transverse tubule (T-tubule) biogenesis and excitationcontraction coupling. This developmental step structurally linkselectrical excitation to the internal contractile system by providingclose apposition of DHPR located in the T-tubule and RyR located in thesarcoplasmic reticulum. Finally, a properly functioning skeletal musclemust express the appropriate MHC proteins required for the task it mustperform. For example, different muscle fibers express different MHCproteins depending on the rate of contraction and force generationrequired by the work to be done. Consequently, skeletal muscle fiberschange their MHC expression profiles to best meet the requirements ofthe body as it matures. Without these modifications, an in vitro modelof skeletal muscle maturation cannot achieve full physiologicalrelevance.

One approach for identifying the role of specific growth factors andhormones in muscle differentiation is to develop an in vitro modelsystem consisting of a serum-free medium supplemented with the factorsof interest. Such a model provides the opportunity to evaluate the roleof each factor individually or in combination with others known orbelieved to be important in skeletal muscle development. For example,the concentration and/or temporal application of medium components inorder to influence the maturation of extrafusal fiber or intrafusalfiber subtypes could be easily investigated.

Employing a non-biological growth substrate such astrimethoxy-silylpropyl-diethylenetriamine (DETA) provides an additionalmeasure of control. DETA is a silane molecule that forms a covalentlybonded monolayer on glass coverslips, resulting in a uniform,non-hydrophilic surface for cell growth. The use of DETA surfaces isadvantageous from a tissue engineering perspective because it can becovalently linked to virtually any hydroxolated surface, it is amenableto patterning using standard photolithography and it promotes long-termcell survival because it is non-digestible by matrix metalloproteinasessecreted by the cells [24, 25].

Previously, studies have demonstrated the usefulness of the DETA silanesubstrate for in vitro culture systems. Interesting features of the DETAsilane are that its molecular geometry does not allow for an orderednanolayer and may partially mimic the three dimensional features of anextracellular matrix, which may be responsible for robust growth ofdifferent cell types on this synthetic substrate [24-31]. Additionally,DETA's non-biological nature supports the analysis of ECM proteinssecreted by the cell in response to different in vitro conditions.

We earlier developed a defined system that promoted differentiation ofdifferent skeletal muscle phenotypes and resulted in the formation ofcontractile myotubes. This resulted in short-term survival of themyotubes [25, 28]. We also have developed a novel bio-hybrid technologyto integrate functional myotubes with cantilever based bio-MEMS devicesfor the study of muscle physiology, neuromuscular junction formation andbio-robotics applications for use in a model of the stretch reflex arc[32]. More recently, using our defined model system, we have achieved asignificant breakthrough by creating mechanosensitive intrafusalmyotubes in vitro [33]. The intrafusal fibers differentiated uponaddition of neuregulin 1-β-1 to serum-free medium in our defined system.Intrafusal fibers are the myotubes present in the muscle spindle whichfunctions as the sensory receptor of the stretch reflex circuit [16] andcombined with extrafusile fibers represent the primary componentnecessary to reproduce functional muscle function in vitro.

This system has been utilized as a model for different developmental andfunctional applications, however, further improvements are necessary toenhance the physiological relevance of the skeletal muscle myotubes [32,33]. Specifically, in order to create a working model of the stretchreflex arc, myotubes are needed that more accurately representextrafusal fibers in vivo. A more advanced developmental system forskeletal muscle would have applications in basic science research andtissue engineering. In this study, we have demonstrated sarcomereassembly, the development of the excitation-contraction couplingapparatus and myosin heavy chain (MHC) class switching.

The results disclosed herein suggest we have discovered a group ofbiomolecules that act together as a molecular switch promoting thetransition from embryonic to neonatal MHC expression as well as otherstructural adaptations resulting in the maturation of skeletal muscle invitro. The discovery of these biomolecular switches will be a powerfultool in regenerative medicine and tissue engineering applications suchas skeletal muscle tissue grafts. It should also be useful in highercontent high-throughput screening technology.

SUMMARY OF THE INVENTION

With the foregoing in mind, the present invention advantageouslyprovides a method of culturing mammalian muscle cells. The method of theinvention includes preparing one or more carriers coated with acovalently bonded monolayer of trimethoxy-silylpropyl-diethylenetriamine(DETA). This is followed by verifying DETA monolayer formation by one ormore associated optical parameters. The method continues by suspendingisolated fetal rat skeletal muscle cells in serum-free medium accordingto medium composition 1, as set forth below in further detail, thenplating the suspended cells onto the prepared carriers at apredetermined density. The method then calls for leaving the carriersundisturbed while the plated cells adhere to the DETA monolayer andcovering the carriers with a mixture of medium composition 1 and mediumcomposition 2, both as described below. Finally, the method ends byincubating the carriers.

In the method, the one or more carriers typically comprise glass coverslips. Those of skill in the art should understand that verifying isaccomplished by an optical contact angle goniometer in the presentinvention, which may also include verifying by X-ray photoelectronspectroscopy (XPS). In the method, verifying may be accomplished by bothan optical contact angle goniometer and by XPS.

Our current cantilever system is designed for force measurements ofcontracting muscle cells and uses laser optics as a readout system[136]. Alternatively, piezoresistive and piezoelectric approaches arethe most widely applied techniques for measuring stress applied onmicrocantilevers [137] and could be easily adapted to the presentinvention by those of ordinary skill in the art. The advantage is thatthe mechanical device and the read our electronics can be implemented inthe same integrated circuit. Replacing the optical readout withpiezoelements will reduce the size and complexity of our currentcantilever system.

Those skilled in the art will know that piezoelectricity is the abilityof certain materials (crystals and certain ceramics) to generate anelectric potential in response to applied mechanical stress [132]. Thepiezoelectric effect is used in various sensors to measure stresses orgeometrical deformations in various mechanical devices. The reversepiezoelectric effect turns piezoelectric materials into actuators, whenan external voltage is applied to the crystal [133]. Piezoelectricmaterials are e.g. quartz, bone, sodium tungstate, zinc oxide, or leadzirconate titanate (PZT) [134]. A similar effect is the piezoresistivephenomenon. When subjected to mechanical stress, these materials changetheir resistivity [135].

Culture aspects of the method include wherein plating the muscle cellsis at a density of approximately from 700 to 1000 cells/mm2. Then,leaving the carriers undisturbed continues for approximately one hourand incubating is effected under physiologic conditions and may best beaccomplished at approximately 37° C. in an air atmosphere with about 5%CO₂ and 85% humidity. The culture is then covered with a mixture ofapproximately equal volumes of medium composition 1 and mediumcomposition 2. Preferably, an initial complete change of the mediumcovering the carriers is accomplished by substituting NBactiv4® mediumduring incubation. Moreover, after the initial complete change ofmedium, changing every three days more than half of the medium coveringthe carriers is preferred and it is most preferred changing every threedays approximately three quarters of the medium covering the carriers.

Another embodiment of the present invention includes a method ofculturing mammalian muscle cells which comprises allowing mammalianfetal muscle cells suspended in medium according to composition 1toadhere to a monolayer of covalently bonded DETA formed on an underlyingcarrier surface and incubating the adhered cells covered in a mixture ofapproximately equal volumes of medium composition 1 and mediumcomposition 2.

In the methods of the invention, the mammalian fetal muscle cells maycomprise fetal rat cells and the underlying carrier surface may comprisea glass cover slip. Incubating is preferably under physiologicalconditions, typically at approximately 37° C. in an atmosphere of airwith about 5% CO₂ and 85% humidity.

In the alternate embodiment of the invention, the method includeschanging the covering medium to NBactiv4®, preferably afterapproximately four days of incubation. Thereafter, the method calls forchanging every three days more than half of the medium covering thecarriers and preferably about three quarters of the medium covering thecarriers.

Also part of the invention is a new cell culture medium compositionwhich includes NBactiv4®, an antibiotic-antimycotic composition,cholesterol, human TNF-alpha, PDGF BB, vasoactive intestinal peptides,insulin-like growth factor 1, NAP, r-Apolipoprotein E2, purified mouseLaminin, beta amyloid, human tenascin-C protein, rr-Sonic hedgehog ShhN-terminal, and rr-Agrin C terminal. This medium composition may beamplified with G5 supplement, VEGF, acidic fibroblast growth factor,heparin sulphate, LIF, rat plasma Vitronectin, CNTF, GNDF, NT-3, NT-4,BDNF and CT-1.

BRIEF DESCRIPTION OF THE DRAWINGS

Some of the features, advantages, and benefits of the present inventionhaving been stated, others will become apparent as the descriptionproceeds when taken in conjunction with the accompanying drawings,presented for solely for exemplary purposes and not with intent to limitthe invention thereto, and in which:

FIG. 1. is a schematic diagram of a culture protocol according to anembodiment of the present invention;

FIG. 2 A, B, C and D provide phase pictures of 50 day old myotubes inculture; red arrows showing characteristic striations in most of themyotubes; scale bar equals 75 μm;

FIG. 3 shows myotubes stained with antibodies against embryonic myosinheavy chain (F 1.652) proteins at day 50; scale bar is 75 μm; A) panelshowing phase+fluorescence picture of the myotubes; B) another view ofpanel A but observed only under fluorescence; white arrows showing thestriations; C) panel showing image of myotubes under phase+fluorescenceillumination; D) shows panel C observed only under fluorescenceillumination; E) panel showing phase+fluorescence picture of themyotubes (white arrow indicating the striations); F) panel E observedonly in fluorescence light (white arrow indicating the striations); G)panel showing phase+fluorescence picture of the myotubes (white arrowindicating the striations); H) panel G observed only under fluorescencelight (white arrow indicating the striations);

FIG. 4 shows myotubes immunostained with neonatal myosin heavy chain(N3.36) and alpha-bungarotoxin at day 50; scale bar is 75 μm; A) is aphase picture of 2 myotubes indicated by white arrows; B) both themyotubes shown in phase (panel A) have acetylcholine receptor clusteringindicated by alpha-bungarotoxin staining; C) only one myotube out of thetwo seen in panel A stained for N3.36; D) double stained image of panelA with alpha-bungarotoxin and N3.36; E) phase image of 6 myotubesindicated by white arrows; F) all the myotubes shown in phase (panel E)have acetylcholine receptor clustering shown by alpha-bungarotoxinstaining; G) none of the myotubes in panel E stained for N3.36; H) I)and J) show differential staining of the myotubes with N3.36; K) L) andM) showing differential staining of the myotubes with N3.36;

FIG. 5 illustrates ryanodine receptor and DHPR receptor clustering in 30days old skeletal muscle culture (scale bar 75 μm); A) phase andfluorescent-labeled picture of the myotubes; B) merged fluorescencepicture of the ryanodine receptor (green) and DHPR receptor (red)clustering on the myotubes shown in panel A; C) ryanodine receptor(green) on the myotubes shown in panel A; D) DHPR receptors on themyotubes shown in panel A; E) phase and fluorescent-labeled picture ofthe myotubes; F) merged fluorescent picture of the ryanodine receptor(green) and DHPR receptor (red) clustering on the myotubes (panel E); G)ryanodine receptor (green) on the myotubes (panel E); H) DHPR receptorson the myotubes (panel E); I) phase and fluorescent-labeled picture ofthe myotubes; J) K) and L) show merged fluorescence pictures of theryanodine receptor (green) and DHPR receptor (red) clustering on themyotubes (panel I) at three different planes (white arrows indicate thestriations and the receptor clustering);

FIG. 6 shows ryanodine receptor and DHPR receptor clustering in 100 daysold skeletal muscle culture (scale bar: 75 μm); A) shows phase andfluorescent-labeled picture of the myotubes; B) is a merged fluorescencepicture of the ryanodine receptor (green) and DHPR receptor (Red)clustering on the myotubes (panel A); C) shows ryanodine receptor(green) on the myotubes (panel A); D) shows DHPR receptors on themyotubes (panel A); E and F show views of the same panels at differentplanes showing the merged fluorescent picture of the ryanodine receptor(green) and DHPR receptor (red) clustering on the myotubes;

FIG. 7 depicts patch clamp electrophysiology of the myotubes, wherein Ashows representative voltage clamp trace obtained after patching a 48days old myotube in culture; B shows representative current clamp traceof the same myotube for which voltage clamp trace had been obtained(inset showing the picture of patched myotubes).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention will now be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionpertains. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below. Anypublications, patent applications, patents, or other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including any definitions,will control. In addition, the materials, methods and examples given areillustrative in nature only and not intended to be limiting.Accordingly, this invention may, however, be embodied in many differentforms and should not be construed as limited to the illustratedembodiments set forth herein. Rather, these illustrated embodiments areprovided so that this disclosure will be thorough and complete, and willfully convey the scope of the invention to those skilled in the art.Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

Materials and Methods

Surface Modification and Characterization

Glass coverslips (Thomas Scientific 6661F52, 22×22 mm No. 1) werecleaned using an O₂ plasma cleaner (Harrick PDC-32G) for 20 minutes at100 mTorr. The DETA (United Chemical Technologies Inc. T2910KG) filmswere formed by the reaction of the cleaned glass surface with a 0.1%(v/v) mixture of the organosilane in freshly distilled toluene (FisherT2904). The DETA coated coverslips were then heated to approximately QQ100° C., rinsed with toluene, reheated to approximately 100° C., andthen oven dried [28]. Surfaces were characterized by contact anglemeasurements using an optical contact angle goniometer (KSV Instruments,Cam 200) and by X-ray photoelectron spectroscopy (XPS) (Kratos Axis165). XPS survey scans, as well as high-resolution N1s and C1s scansutilizing monochromatic Al Kα excitation were obtained [28].

Skeletal Muscle Culture and Serum Free Medium

The skeletal muscle was dissected from the thighs of the hind limbs offetal rats (17-18 days old). The tissue was collected in a sterile 15 mLcentrifuge tube containing 1 mL of phosphate-buffered saline (calcium-and magnesium- free) (Gibco 14200075). The tissue was enzymaticallydissociated using 2 mL of 0.05% of trypsin-EDTA (Gibco 25300054)solution for 30 minutes in a 37° C. water bath at 50 rpm. After 30minutes the trypsin solution was removed and 4 mL of Hibernate E +10%fetal bovine serum (Gibco 16000044) was added to terminate the trypsinreaction. The tissue was then mechanically triturated with thesupernatant being transferred to a 15 mL centrifuge tube. The sameprocess was repeated two times by adding 2 mL of L15 +10% FBS each time.The 6 mL cell suspension obtained after mechanical trituration wassuspended on a 2 mL, 4% BSA (Sigma A3059) (prepared in L15 medium)cushion and centrifuged at 300g for 10 minutes at 4° C. The pelletobtained was washed 5 times with L15 medium then resuspended in 10 mL ofL15 and plated in 100mm uncoated dishes for 30 minutes. The non-attachedcells were removed and then centrifuged on a 4% BSA cushion [28]. Thepellet was resuspended in serum-free medium according to the protocolillustrated in FIG. 1 and plated on the coverslips at a density of700-1000 cells/mm². The serum-free medium containing different growthfactors and hormones was added to the culture dish after one hour. Thefinal medium was prepared by mixing medium one (Table 1) and medium two(Table 2) in a 1:1 v/v ratio. FIG. 1 indicates the flowchart of theculture protocol. Tables 1 and 2 indicated the growth factor and hormonesupplement compositions of medium one and medium two. The cells weremaintained in a 5% CO₂ incubator (relative humidity 85%). The fullmedium was replaced after four days with NBactiv4® medium according tothe protocol in FIG. 1 [34]. Thereafter three-fourth of the medium waschanged every three days with NBactiv4®.

NbActiv4® (available from BrainBits LLC) comprises all of theingredients in Neurobasal™, B-27™, and GlutaMAX™. NbActiv4® alsocomprises creatine, estrogen, and cholesterol.

Immunocytochemistry of Skeletal Muscle Myotubes

Coverslips were prepared for immunocytochemical analysis as previouslydescribed. Briefly, coverslips were rinsed with PBS, fixed in −20□ Cmethanol for 5-7 min, washed in PBS, incubated in PBS supplemented with1% BSA and 0.05% saponin (permeabilization solution) for 10 minutes, andblocked for 2 h with 10% goat serum and 1% BSA. Cells were incubatedovernight with primary antibodies against embryonic myosin heavy chain(F1.652) (dilution>1:5), neonatal myosin heavy chain (N3.36) (1:5)(Developmental Studies Hybridoma Bank), ryanodine receptor (AB9078,Millipore) (1:500) and dihydropyridine binding complex (α1-Subunit) (MAB4270, Millipore) (1:500) diluted in the blocking solution. Cells werewashed with PBS and incubated with the appropriate secondary antibodiesfor two hours in PBS. After two hours the coverslips were rinsed withPBS and mounted on glass slides and evaluated using confocal microscopy[25, 28, 31].

AChR Labeling of Myotubes

AChRs were labeled as described previously by incubating cultures with5×10-8 M of α-bungarotoxin, Alexa Fluor® 488 conjugate (B-13422;Invitrogen) for 1.5 h at 37° C. [12, 31]. Following incubation inα-bungarotoxin, the cultures were fixed as above for subsequent stainingwith embryonic myosin heavy chain (F1.652) antibodies.

Patch Clamp Electrophysiology of the Myotubes

Whole-cell patch clamp recordings were performed in a recording chamberlocated on the stage of a Zeiss Axioscope 2FS Plus upright microscope asdescribed previously [25, 33]. The chamber was continuously perfused (2ml/min) with the extracellular solution (Leibovitz medium, 35° C.).Patch pipettes were prepared from borosilicate glass (BF150-86-10;Sutter, Novato, Calif.) with a Sutter P97 pipette puller and filled withintracellular solution (K-gluconate 140 mM, EGTA 1 mM, MgCl₂ 2 mM,Na₂ATP 2 mM, phosphocreatine 5 mM, phosphocreatine kinase 2.4 mM, Hepes10 mM; pH=7.2). The resistance of the electrodes was 6-8MΩ. Voltageclamp and current clamp experiments were performed with a Multiclamp700A amplifier (Axon Laboratories, Union City, Calif.). Signals werefiltered at 2 kHz and digitized at 20 kHz with an Axon Digidata 1322Ainterface. Data recording and analysis were done with pClamp 8 software(Axon Laboratories). Membrane potentials were corrected by subtractionof a 15 mV tip potential, which was calculated using Axon's pClamp 8program. Sodium and potassium currents were measured in voltage clampmode using voltage steps from a −85 mV holding potential. Actionpotentials were evoked with 1 second depolarizing current injectionsfrom a -85 mV holding potential [25, 28].

Results

DETA Surface Modification and Characterization

Static contact angle and XPS analysis was used for the validation of thesurface modifications and for monitoring the quality of the surfaces.Stable contact angles (40.64±2.9/mean±SD) throughout the study indicatedhigh reproducibility and quality of the DETA surfaces and were similarto previously published results [24, 25, 28, 29, 31]. Based on the ratioof the N (401 and 399 eV) and the Si 2p3/2 peaks, XPS measurementsindicated that a reaction-site limited monolayer of DETA was formed onthe coverslips [35].

Development of the Serum Free Medium Formulation and Culture Timelinefor Long-Term Survival and Maturation of Myotubes

The serum free medium composition was developed empirically. The finalmedium is derived from two different medium compositions described inTables 1 and 2. Table 1 constitutes the same medium composition usedpreviously for a motoneuron-muscle co-culture and adult spinal cordneurons culture [26, 27, 30, 31]. Table 2 is composed of twelveadditional factors that had been shown to promote skeletal musclematuration and neuromuscular junction formation separately. The finalmedium was prepared by mixing these two media in a 1:1 v/v ratio. Afterfirst 4 days of culture the whole medium was replaced with NBactiv4®medium [34]. hereafter, every three days three-fourth medium was changedwith NBactiv4®. The culture technique has been illustrated in theflowchart (FIG. 1).

Using this new medium formulation and timeline, myotubes weresuccessfully cultured for more than 50 days. FIG. 2 indicates 50 daysold myotubes in culture. As the myotubes aged and grew they began toform the characteristic anisotropic (A band) and isotropic (I band)banding pattern observed with in vivo muscle fibers [22, 23]. Thisbanding pattern is caused by differential light diffraction due to theorganization of myofibril proteins forming sarcomeres within themyotubes [22, 23]. The arrowheads in the images (FIG. 2A-D) indicatemyotubes where sarcomeric organization has occurred and is visualized bythe appearance of A and I bands.

Myotube Expression of Fetal Myosin Heavy Chain

The myotubes formed were evaluated for the expression of fetal MHC toestablish a baseline as comparison to our previous results [28]. In FIG.3, the myotubes phenotypes formed at approximately day 50 in vitro areshown. The myotubes ranged from having clustered nuclei (FIG. 3A-D) tohaving diffuse nuclear organization (FIG. 3E-H). The arrowheads in theimages indicate the characteristic striations.

Differential Expression of Neonatal MHC Protein in the Myotubes

In order to determine if the myotubes were maturing in a physiologicallyrelevant way as they aged in vitro, the expression of neonatal MHCprotein was evaluated. After approximately 50 days in vitro 25% of themyotubes expressed neonatal MHC (FIG. 4A-M). Additionally, the myotubeswere stained for clustering of acetylcholine receptors (AChR) usingalpha bungarotoxin (FIG. 5B,F). This clustering of the AChR receptors,induced by the motoneuron protein agrin in vivo, are locations on themyotube where neuromuscular junction formation occurs.

Formation of the Excitation—Contraction Coupling Apparatus

The presence of ryanodine (RyR) receptor and dihydropyridine (DHPR)receptor clusters, as well as their colocalization in vivo, representsthe development of excitation-contraction coupling apparatus in skeletalmuscle myotubes [19, 21-23]. The clustering of both RyR and DHPRreceptors was observed on the myotubes after 30 days in culture (FIG.5A-D). The clustering and colocalization of the RyR+DHPR clusters wasobserved with different myotube morphologies (FIG. 5E-L). Thisfunctional adaptation illustrated that the medium formulationfacilitated not only the structural maturation but also the functionalmaturation of myotubes in this in vitro system. The clustering of theRyR+DHPR receptors was also observed in the 70 day old myotubes,indicating that the older myotubes maintained their functional integrity(FIG. 6A-F).

Myotube Electrophysiology

The myotubes contracted spontaneously in the culture and thecontractions began generally by day four and continued throughout thelife of the culture. Most of the myotubes expressed functional voltagegated sodium, potassium and calcium ion channels as reported previously[28]. The voltage clamp electrophysiology of the myotubes indicated theinward and outward currents that demonstrate functional sodium andpotassium channels (FIG. 7A). The current clamp study indicated thesingle action potential fired by the myotubes (FIG. 7B).

Discussion

Herein we have documented the development of a system for long-term invitro functional, skeletal muscle culture. This system was developed inresponse to a need for more physiologically relevant skeletal musclemyotubes for functional in vitro systems. For our specific research,they were needed for a realistic model of the stretch reflex arcdevelopment and to be integrated with bio-MEMS cantilevers for screeningappications. The results indicate we achieved three significantstructural modifications within the myotubes, causing both thedevelopmental profile and functionality of the fibers to better mimic invivo physiology. It is believed that this skeletal muscle maturationresulted from modifications to the cell culture technique, a new mediumformulation and the use of NBactiv4® as the maintenance medium.

The presently described serum-free medium supplemented with growthfactors was developed to support the survival, proliferation and fusionof fetal rat myoblasts into contractile myotubes. The rationale forselecting the growth factors was based on the distribution of theircognate receptors in the developing myotubes in rat fetus [1-11]. Tables1 and 2 reference the literature where these individual growth factors,hormones and neurotransmitters were observed to support muscle andneuromuscular junction development. The composition in Table 1 is theformulation used for a previously published medium used formotoneuron-muscle co-culture and adult spinal cord neuron culture [26,27, 30, 31]. Table 2 lists the twelve additional factors we haveidentified in muscle development and neuromuscular junction formation.The use of NBactiv4® for the maintenance of the cells providedunexpected results in that it significantly improved the survival of theskeletal muscle derived myotubes despite the original development ofNBactiv4® for the long-term maintenance and synaptic connectivity offetal hippocampal neurons in vitro [34].

We observed a ratio of 25% neonatal to 75% embryonic MHC expression ofthe myotubes, which contrasts with the previous study in which MHCexpression was strictly embryonic. We believe that the myotubes maturedin this culture system because the long-term survival provided adequatetime for the myotubes to respond to the additional growth factors, whichactivated the necessary signaling pathways to achieve MHC classswitching [20]. This suggests that a different growth factor profilecould be utilized to activate alternative signaling pathways and drivemyotube differentiation down other pathways. For example, the effects ofadding steroid hormones like testosterone to the system could becritically examined.

The colocalization of RyR and DHPR clusters in the myotubes indicatedthe formation of excitation-contraction coupling apparatus and wasanother indicator of functional maturation in the fibers.Excitation-contraction coupling is the signaling process in muscle bywhich membrane depolarization causes a rapid elevation of the cytosolicCa²⁺ generating contractile force [36]. The close proximity of the DHPRand RyR complexes occurs at specialized junctions established betweenthe transverse tubule and sarcoplasmic reticulum (SR) membranes inskeletal muscle myotubes [37]. At these junctions, T-tubuledepolarization is coupled to Ca²⁺ release from the SR resulting inmuscle contraction [38-40]. This structural adaptation represents asignificant functional change due to the fact thatexcitation-contraction coupling is required for successful extrafusalmuscle fiber development as well as neuromuscular junction formation[19, 21-23]. This improved model provides the potential to studyexcitation-contraction coupling in a defined system as well as myotonicand myasthenic diseases.

Conclusion

The development of sarcomeric structures, the excitation-contractioncoupling apparatus and MHC class switching in the skeletal musclemyotubes is a result of the improvements to the model system documentedin this research. This improved system along with the new findingssupport the goal of creating physiologically relevant tissue engineeredmuscle constructs and puts within reach the goal of functional skeletalmuscle grafts. Furthermore, we believe this serum-free culture systemwill be a powerful tool in developing advanced strategies forregenerative medicine in muscular dystrophies, stretch reflex arcdevelopment and integrating skeletal muscle with bio-hybrid prostheticdevices.

Accordingly, in the drawings and specification there have been disclosedtypical preferred embodiments of the invention and although specificterms may have been employed, the terms are used in a descriptive senseonly and not for purposes of limitation. The invention has beendescribed in considerable detail with specific reference to theseillustrated embodiments. It will be apparent, however, that variousmodifications and changes can be made within the spirit and scope of theinvention as described in the foregoing specification and as defined inthe appended claims.

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TABLE 1 Medium Composition 1 S. Cata- Refer- No Component Amount logue #Source ences 1. Neurobasal 500 ml 10888 Gibco/ [41] Invitrogen 2.Antibiotic- 5 ml 15240-062 Gibco/ Antimycotic Invitrogen 3. G5Supplement 5 ml 17503-012 Gibco/ [42-51] (100X) Invitrogen 4.VEGF_(165 r Human) 10 μg P2654 Gibco/ [52-55] Invitrogen 5. Acidic FGF12.5 μg 13241-013 Gibco/ [42, 49, Invitrogen 51, 56-61] 6. HeparinSulfate 50 μg D9809 Sigma [42, 49, 51, 56-61] 7. LIF 10 μg L5158 Sigma[62-70] 8. Vitronectin 50 μg V0132 Sigma [71, 72] (Rat Plasma) 9. CNTF20 μg CRC 401B Cell [73-77] Sciences 10. NT-3 10 μg CRN 500B Cell [15]Sciences 11. NT-4 10 μg CRN 501B Cell [78, 79] Sciences 12. GDNF 10 μgCRG 400B Cell [80-84] Sciences 13. BDNF 10 μg CRB 600B Cell [79, 85,Sciences 86] 14. CT-1 10 μg CRC 700B Cell [87-95] Sciences

TABLE 2 Medium Composition 2 Refer- No Component(s) Amount CatalogSource ences 1 Neurobasal 500 ml 10888 Invitrogen/ [41] Gibco 2Antibiotic- 5 ml 15240- Invitrogen/ antimycotic 062 Gibco 3 Cholesterol5 ml 12531 Invitrogen/ [96] (250X) Gibco 4 TNF-alpha, 10 μg T6674 Sigma-[97-99] human Aldrich 5 PDGF BB 50 μg P4056 Sigma- [62, 100- Aldrich103] 6 Vasoactive 250 μg V6130 Sigma- [104] intestinal Aldrich peptide(VIP) 7 Insulin-like 25 μg I2656 Sigma- [68, 69, 98] growth Aldrichfactor 1 8 NAP 1 mg 61170 AnaSpec, [105, 106] Inc. 9 r- 50 μg P2002Panvera, [107] Apolipoprotein Madison, WI E2 10 Laminin, 2 mg 08-125Millipore [108-114] mouse purified 11 Beta amyloid 1 mg AG966 Millipore[115-117] (1-40) 12 Human 100 μg CC065 Millipore [118] Tenascin- Cprotein 13 rr-Sonic 50 μg 1314-SH R&D [7, 119-129] hedgehog, Systems ShhN-terminal 14 rr-Agrin 50 μg 550-AG- R&D [130, 131] (C terminal) 100Systems

TABLE 3 B-27 ™ Serum-Free Supplement Media ingredients ConcentrationComponents (mg/L) Vitamins Biotin 0.10 DL Alpha Tocopherol Acetate 1.0DL Alpha-Tocopherol 1.0 Vitamin A 0.1-0.2 Proteins BSA, fatty acid freeFraction V 2500.0 Catalase 2.5 Human Recombinant Insulin 4.0 HumanTransferrin 5.0 Superoxide Dismutase 2.5 Other Components Corticosterone0.02 D-Galactose 15.0 Ethanolamine HCl 1.0 Glutathione (reduced) 1.0L-Carnitine HCl 2.0 Linoleic Acid 1.0 Linolenic Acid 1.0 Progesterone0.0063 Putrescine 2HCl 16.1 Sodium Selenite 0.035 T3(triodo-I-thyronine) 0.002

TABLE 4 Neurobasal ™ media formulation Concentration Components (mg/L)Amino Acids Glycine 30.0 L-Alanine 2.0 L-Arginine hydrochloride 84.0L-Asparagine-H2O 0.83 L-Cysteine 31.5 L-Histidine hydrochloride-H2O 42.0L-Isoleucine 105.0 L-Leucine 105.0 L-Lysine hydrochloride 146.0L-Methionine 30.0 L-Phenylalanine 66.0 L-Proline 7.76 L-Serine 42.0L-Threonine 95.0 L-Tryptophan 16.0 L-Tyrosine 72.0 L-Valine 94.0Vitamins Choline chloride 4.0 D-Calcium pantothenate 4.0 Folic Acid 4.0Niacinamide 4.0 Pyridoxal hydrochloride 4.0 Riboflavin 0.4 Thiaminehydrochloride 4.0 Vitamin B12 0.0068 i-Inositol 7.2 Inorganic SaltsCalcium. Chloride (CaCl2) (anhyd.) 200.0 Ferric Nitrate (Fe(NO3)3″9H2O)0.1 Magnesium Chloride (anhydrous) 77.3 Potassium Chloride (KCl) 400.0Sodium Bicarbonate (NaHCO3) 2200.0 Sodium Chloride (NaCl) 3000.0 SodiumPhosphate monobasic 125.0 (NaH2PO4—H2O) Zinc sulfate (ZnSO4—7H2O) 0.194Other D-Glucose (Dextrose) 4500.0 Components HEPES 2600.0 Phenol Red 8.1Sodium Pyruvate 25.0

TABLE 5 Glutamax ™ media formulation Components Concentration (mM)Peptides L-alanyl-L-glutamine 200 Inorganic Salts Sodium Chloride (NaCl)145

That which is claimed:
 1. A method of maintaining a muscle cell culture,the method comprising: maintaining the muscle cell culture in aserum-free maintenance medium comprising the components at theconcentrations listed in Table 3, the components at the concentrationslisted in Table 4, the components at the concentrations listed in Table5, creatine, estrogen, and cholesterol wherein the cells in the musclecell culture consist of muscle cells.
 2. The method of claim 1, whereinthe muscle cell culture is maintained in the serum-free maintenancemedium for at least 30 days.
 3. The method of claim 2, wherein themuscle cell culture is maintained in the serum-free maintenance mediumfor at least 50 days.
 4. The method of claim 1, further comprisingplating the muscle cell culture onto a non-biological growth substrateprior to maintaining the muscle cell culture in the serum-freemaintenance medium.
 5. The method of claim 4, wherein the non-biologicalgrowth substrate comprises a silane molecule.
 6. The method of claim 5,wherein the non-biological growth substrate istrimethoxy-silylpropyl-diethylenetriamine (DETA).
 7. The method of claim1, further comprising plating the muscle cell culture at a density of700 to 1000 muscle cells per square millimeter prior to maintaining themuscle cell culture in the serum-free maintenance medium.
 8. The methodof claim 1, further comprising replenishing the serum-free maintenancemedium every 1 to 5 days.
 9. The method of claim 8, further comprisingreplenishing the serum-free maintenance medium every 3 days.
 10. Themethod of claim 1, further comprising incubating the muscle cell culturein a first serum-free medium prior to maintaining the muscle cellculture in the serum-free maintenance medium.
 11. The method of claim10, wherein the first serum-free medium comprises a factor selected fromthe group consisting of vascular endothelial growth factor (VEGF),acidic fibroblast growth factor (FGF), heparin sulfate, leukemiainhibitory factor (LIF), vitronectin, ciliary neurotrophic factor(CNTF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), glial cell-derivedneurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF),cardiotrophin-1 (CT-1), cholesterol, tumor necrosis factor alpha(TNF-alpha), platelet derived growth factor (PDGF), vasoactiveintestinal peptide, insulin-like growth factor 1, neutrophil activatingprotein (NAP), r-Apolipoprotein, laminin, beta amyloid, tenascin-Cprotein, rr-sonic hedgehog, and rr-agrin.
 12. The method of claim 11,wherein the first serum-free medium comprises vitronectin.
 13. Themethod of claim 12, wherein the first serum-free medium is a mixture ofapproximately equal volumes of the medium composition of Table 1 and themedium composition of Table
 2. 14. The method of claim 10, wherein themuscle cell culture is maintained in the first serum-free medium from 2to 6 days.
 15. The method of claim 14, wherein the muscle cell cultureis maintained in the first serum-free medium for 4 days.